Sperm acquire motility and the ability to fertilize and egg during their transit in the epididymis; post testicular sperm maturation and storage involve mechanisms mediated by the epididymal mucosa. One of the most intriguing and understudied aspects of male reproductive physiology is the ability of the excurrent duct to prevent the development of autoimmune responses against millions of autoantigenic spermatogenic cells and mature spermatozoa, meanwhile initiating very efficient immune responses against pathogenic microorganisms. The present application is focused on the role of macrophages (M?) and dendritic cells (DCs) in establishing the protective environment in which spermatozoa mature and are stored. M? and DCs are two closely related and extremely versatile families of cells that play critical roles in the initiation and regulation of immune responses. We have previously shown that the epididymis is populated by a dense and heterogeneous network of mononuclear phagocytes (MPs) expressing M? and DC markers; in the proximal epididymis, they establish intimate interactions with epididymal epithelial cells and are part of the blood- epididymis barrier. Therefore, they are strategically positioned to play a major role in the contro of male reproductive function. We hypothesize that M? and DCs cooperate to survey both sides of the blood- epididymis barrier to regulate the balance between immune tolerance and inflammation in the post-testicular environment. In Specific Aim 1, we characterize luminal antigen uptake and elucidate the mechanisms underlying tolerance to sperm antigens via the induction of a specific subset of regulatory T lymphocytes (Tregs). In Specific Aim 2, we will determine how M? and DCs prevent circulating pathogens from affecting spermatozoa by surveying local capillaries. Both aims will rely on the use of state-of-the-art microscopy imaging procedures (3D laser scanning confocal microscopy and two-photo intravital microscopy), transgenic mouse models, and immunological techniques (e.g. flow cytometry analysis, cross-talk assays to characterize MPs and T cell interactions) rarely used in reproductive physiology. The ultimate goal of this project is to provide new frameworks for the treatment of male immunological infertility (a major public health issue) and, conversely, for the control of male fertility via immunocontraception.